There is a need for safe and efficient vectors for delivery of bioactive agents, particularly DNA. For example, the conventional approach of viral-mediated delivery of DNA has drawbacks, in that the virus may elicit an immune response in the patient or increase the risk of cancer for the patient.
A number of cationic polymers have been investigated for the delivery of bioactive agents, particularly DNA. Poly(ethylenimine) (PEI) and poly(lysine) have been widely studied as DNA condensing agents and transfection vectors and are the standards to which new polymeric vectors are often compared. However, these polymers are also associated with significant levels of cytotoxicity, and high levels of gene expression are usually realized only at a substantial cost to cell viability (see Lynn and Langer (2000) J. Am. Chem. Soc., vol.122, 10761-10768). Further, PEI is not biodegradable and may not be safe for long-term treatment of a patient.
Poly(amino ester)s are attractive candidates for use as vectors for DNA delivery, in part because the y are biodegradable and cationic in physiological solutions. There have been some reports of initial investigations into synthesis and characterization of poly(amino ester)s as vectors for transfecting cells with DNA. Lynn and Langer report the synthesis of three types of poly(amino ester)s, all of which have tertiary amine linkages in the polymer backbone, are capable of binding DNA and have low cytotoxicity. However, two of these polymers are soluble only at reduced pH, i.e. not in the physiological range. Further, the reference provides no transfection assay results to show that these polymers have utility as vectors for transfecting cells with DNA. Two of these polymers were later reported in Lynn et al. (Lynn et al. (2001) J. Am. Chem. Soc., vol.123, 8155-8156) as not being water-soluble and, as a result, were not tested further ELS candidates for DNA vectors.
Lynn et al. (supra) describe synthesis of a library of 140 poly(amino ester)s that were assayed for DNA-binding and transfection efficiency. Only 70 of these polymers were sufficiently water-soluble to be assayed further, another 14 did not complex DNA efficiently, and of the remaining 56 polymers, only seven polymers showed transfection efficiencies higher than that of naked DNA and three demonstrated cytoxicity. However, the results of Lynn et al. show no clear relationship between chemical structure of the polymer and biological transfection efficiency or other properties.
Lim et al. describe a hyperbranched poly(amino ester) that has transfection efficiency comparable to PEI and low cytotoxicity (Lim et al. (2002) Bioconjugate Chem. vol. 13, 952). The polymer contains both primary amino and tertiary amino functional groups, which groups arm suggested by Lim et al. to be involved in DNA-binding and endosome escape of DNA, respectively. However, the method of making this hyperbranched, multi-functional polymer is inconvenient, as it involves several steps: making an AB2-type monomer by reacting methyl acrylate with tris(hydroxymethyl)aminomethane, polymerizing the AB2-type monomer to obtain a hyperbranched network, then attaching primary amino groups to the branched network.
Many hyperbranched polyesters have poor solubility in water. To address this problem, Gao et al. (Gao et al. (2002) J. Polymer Sci. (Part A) vol.40, 2340-2349) synthesized a series of poly(amino ester)s via the Michael addition of a diamine monomer containing a secondary and a primary amino group to a divinyl monomer. The resulting polymers were reported by Gao et al. to be “hyperbranched”, containing between 58.2% and 75.5% of the repeating units as either being “branched” units (in which all amino groups are tertiary) or “terminal” units (reported as having an unreacted primary amino groups) and were reported to be “quite soluble in water”.
Other types of cationic polymers have been suggested for use as vectors for transfection of cells with DNA, including: polyphosphates (WO 02/092667; Wang et al. (2001) J. Am. Chem. Soc. vol.123, 9480-9481); polyphosphoramidate bearing spermidine side chain (PPA-SP) (WO 03/000776; and Wang et al. (2002) J. Controlled Release vol. 83, 156-168); poly(alpha-(4-aminobutyl)-L-glycolic acid) (PAGA) (U.S. Pat. No. 6,217,912; U.S. Pat. No. 6,267,987B1; WO 01/97781A1; and Lim et al. (2000) J. Am. Chem. Soc. vol. 122, 6524-6525). However, for some of the foregoing polymers, there is no data available to show that the polymers are useful as vectors for transfecting cells with DNA. For those polymers for which transfection data is available, many have drawbacks. For example, transfection efficiencies for some polymers are low relative to PEI and/or high transfection efficiencies are achieved only in the presence of chloroquine. In addition, transfection efficiency can vary depending on the type of cell being transfected, suggesting that there may be a need for a variety of different types of polymers in order to target the various cell types present in the body.